Kimberly D. Brewer

Caspase-responsive smart gadolinium-based contrast agent for magnetic resonance imaging of drug-induced apoptosis

Non-invasive detection of caspase-3/7 activity in vivo has provided invaluable predictive information regarding tumor therapeutic efficacy and anti-tumor drug selection. Although a number of caspase-3/7 targeted fluorescence and positron emission tomography (PET) imaging probes have been developed, there is still a lack of gadolinium (Gd)-based magnetic resonance imaging (MRI) probes that enable high spatial resolution detection of caspase-3/7 activity in vivo. Here we employ a self-assembly approach and develop a caspase-3/7 activatable Gd-based MRI probe for monitoring tumor apoptosis in mice. Upon reduction and caspase-3/7 activation, the caspase-sensitive nano-aggregation MR probe (C-SNAM: 1) undergoes biocompatible intramolecular cyclization and subsequent self-assembly into Gd-nanoparticles (GdNPs). This results in enhanced r1 relaxivity-19.0 (post-activation) vs. 10.2 mM-1 s-1 (pre-activation) at 1 T in solution-and prolonged accumulation in chemotherapy-induced apoptotic cells and tumors that express active caspase-3/7. We demonstrate that C-SNAM reports caspase-3/7 activity by generating a significantly brighter T1-weighted MR signal compared to non-treated tumors following intravenous administration of C-SNAM, providing great potential for high-resolution imaging of tumor apoptosis in vivo.

Optimizing the detection of white matter fMRI using asymmetric spin echo spiral.

The majority of functional magnetic resonance imaging (fMRI) studies restrict their focus to gray matter regions because this tissue is highly perfused relative to white matter. However, an increasing number of studies are reporting fMRI activation in white matter. The current study had two objectives: 1) to evaluate whether it is possible to detect white matter fMRI activation and 2) to determine whether certain MRI contrast mechanisms are more sensitive to white matter activation (i.e., BOLD contrast- versus T(2)-weighting). Data were acquired from a 4 T MRI using an asymmetric spin echo spiral sequence (ASE spiral). This technique collected three images with equal BOLD contrast weighting and increasing T(2)-weighting. An interhemispheric transfer task was used to elicit activation in the corpus callosum. White matter fMRI activation was examined for the averaged ASE spiral data and for each image separately. Callosal activation was present in all subjects as well as in the group analysis. Analyses revealed that increasing T(2) contrast improved sensitivity as measured by percent signal change. The results suggest that it is possible to detect white matter activation in fMRI and that ASE spiral showed increasing sensitivity to this activation as a function of T(2)-weighting. The findings provide further support for the investigation of white matter fMRI.

Functional mapping in the corpus callosum: A 4 T fMRI study of white matter

INTRODUCTION The idea of fMRI activation in white matter (WM) is controversial. Our recent work has used two different approaches to investigate whether there is evidence for WM fMRI. The first approach used words and faces to elicit interhemispheric transfer activation in the posterior corpus callosum (Sperry task). The second approach used checkerboard stimuli to elicit similar activation in the anterior corpus callosum (Poffenberger task). Using these different tasks, it has been possible to detect WM activation in different regions. In the current study, we report the results of a critical experiment: demonstrating that callosal activation can be experimentally manipulated within the same set of individuals. METHODS All subjects completed both the Sperry and Poffenberger tasks. Functional MRI data were acquired at 4T, using an asymmetric spin echo spiral sequence. Data were analyzed with FSL using a model-based approach. Analyses focused on group and individual activations in WM. RESULTS AND DISCUSSION Corpus callosum activation was elicited for both tasks, with activation varying according to task type. A statistical contrast of the two tasks revealed posterior callosal activation for the Sperry task and anterior callosal activation for the Poffenberger task. The Sperry task showed activation in the isthmus and middle body of the corpus callosum at the group level and in 100% of subjects. The Poffenberger task showed activation in the genu and middle body of the corpus callosum at the group level and in 94% of subjects. The WM activation replicated prior results, with the additional strength of functional mapping within the same group of individuals.

Asymmetric spin-echo (ASE) spiral improves BOLD fMRI in inhomogeneous regions.

Functional MRI (fMRI) is of limited use in areas such as the orbitofrontal and inferior temporal lobes due to the presence of local susceptibility‐induced field gradients (SFGs), which result in severe image artifacts. Several techniques have been developed to reduce these artifacts, the most common being the dual‐echo spiral sequences (spiral‐in/out and spiral‐in/in). In this study, a new multiple spiral acquisition technique was developed, in which the later spiral acquisitions are acquired asymmetrically with the peak of a spin‐echo causing increased R2‐weighting but matched R2′‐weighting. This sequence, called asymmetric spin‐echo (ASE) spiral, has demonstrated significant improvements in minimizing the signal loss and increasing the image quality as well as optimal blood‐oxygen‐level‐dependent (BOLD)‐weighting. The ASE spiral is compared to conventional spiral‐out using both signal‐to‐noise ratio (SNR) and whole brain fMRI activation volumes from a breath‐hold task acquired at 4 Tesla. The ASE dual spiral has exhibited SNR increases of up to 300% in areas where strong SFGs are present. As a result, the ASE spiral is highly efficient for recovering lost activation in areas of SFGs, as demonstrated by a 16% increase in the total number of activated voxels over the whole brain. Post spin‐echo ASE spiral images have decreasing SNR due to R2 signal losses, however the increase in R2‐weighting leads to a higher percentage of signal changes producing ASE spiral images with equivalent contrast‐to‐noise ratio (CNR) for each echo. The use of this sequence allows for recovery of BOLD activation in areas of SFG without sacrificing the CNR over the whole brain. Copyright

Molecular Magnetic Resonance Imaging of Tumor Response to Therapy

Personalized cancer medicine requires measurement of therapeutic efficacy as early as possible, which is optimally achieved by three-dimensional imaging given the heterogeneity of cancer. Magnetic resonance imaging (MRI) can obtain images of both anatomy and cellular responses, if acquired with a molecular imaging contrast agent. The poor sensitivity of MRI has limited the development of activatable molecular MR contrast agents. To overcome this limitation of molecular MRI, a novel implementation of our caspase-3-sensitive nanoaggregation MRI (C-SNAM) contrast agent is reported. C-SNAM is triggered to self-assemble into nanoparticles in apoptotic tumor cells, and effectively amplifies molecular level changes through nanoaggregation, enhancing tissue retention and spin-lattice relaxivity. At one-tenth the current clinical dose of contrast agent, and following a single imaging session, C-SNAM MRI accurately measured the response of tumors to either metronomic chemotherapy or radiation therapy, where the degree of signal enhancement is prognostic of long-term therapeutic efficacy. Importantly, C-SNAM is inert to immune activation, permitting radiation therapy monitoring.

Clearance of depot vaccine SPIO-labeled antigen and substrate visualized using MRI

Immunotherapies, including peptide-based vaccines, are a growing area of cancer research, and understanding their mechanism of action is crucial for their continued development and clinical application. Exploring the biodistribution of vaccine components may be key to understanding this action. This work used magnetic resonance imaging (MRI) to characterize the in vivo biodistribution of the antigen and oil substrate of the vaccine delivery system known as DepoVax(TM). DepoVax uses a novel adjuvanted lipid-in-oil based formulation to solubilise antigens and promote a depot effect. In this study, antigen or oil were tagged with superparamagnetic iron oxide (SPIO), making them visible on MR images. This enables tracking of individual vaccine components to determine changes in biodistribution. Mice were injected with SPIO-labeled antigen or SPIO-labeled oil, and imaged to examine clearance of labeled components from the vaccine site. The SPIO-antigen was steadily cleared, with nearly half cleared within two months post-vaccination. In contrast, the SPIO-oil remained relatively unchanged. The biodistribution of the SPIO-antigen component within the vaccine site was heterogeneous, indicating the presence of active clearance mechanisms, rather than passive diffusion or drainage. Mice injected with SPIO-antigen also showed MRI contrast for several weeks post-vaccination in the draining inguinal lymph node. These results indicate that MRI can visualize the in vivo longitudinal biodistribution of vaccine components. The sustained clearance is consistent with antigen up-take and trafficking by immune cells, leading to accumulation in the draining lymph node, which corresponds to the sustained immune responses and reduced tumor burden observed in vaccinated mice.

Using MRI to evaluate and predict therapeutic success from depot-based cancer vaccines.

In the preclinical development of immunotherapy candidates, understanding the mechanism of action and determining biomarkers that accurately characterize the induced host immune responses is critical to improving their clinical interpretation. Magnetic resonance imaging (MRI) was used to evaluate in vivo changes in lymph node size in response to a peptide-based cancer vaccine therapy, formulated using DepoVax (DPX). DPX is a novel adjuvant lipid-in-oil–based formulation that facilitates enhanced immune responses by retaining antigens at the injection site for extended latencies, promoting increased potentiation of immune cells. C57BL/6 mice were implanted with C3 (HPV) tumor cells and received either DPX or control treatments, 5 days post-implantation. Complete tumor eradication occurred in DPX-vaccinated animals and large volumetric increases were observed in the vaccine-draining right inguinal lymph node (VRILN) in DPX mice, likely corresponding to increased localized immune response to the vaccine. Upon evaluating the relative measure of vaccine-potentiated immune activation to tumor-induced immune response (VRILN/VLILN), receiver-operating characteristic (ROC) curves revealed an area under the curve (AUC) of 0.90 (±0.07), indicating high specificity and sensitivity as a predictive biomarker of vaccine efficacy. We have determined that for this tumor model, early MRI lymph node volumetric changes are predictive of depot immunotherapeutic success.

Using lymph node swelling as a potential biomarker for successful vaccination

There is currently a lack of biomarkers to help properly assess novel immunotherapies at both the preclinical and clinical stages of development. Recent work done by our group indicated significant volume changes in the vaccine draining right lymph node (RLN) volumes of mice that had been vaccinated with DepoVaxTM, a lipid-based vaccine platform that was developed to enhance the potency of peptide-based vaccines. These changes in lymph node (LN) volume were unique to vaccinated mice. To better assess the potential of volumetric LN markers for multiple vaccination platforms, we evaluated 100 tumor bearing mice and assessed their response to vaccination with either a DepoVax based vaccine (DPX) or a water–in-oil emulsion (w/o), and compared them to untreated controls. MRI was used to longitudinally monitor LN and tumor volumes weekly over 4 weeks. We then evaluated changes in LN volumes occurring in response to therapy as a potential predictive biomarker for treatment success. We found that for both vaccine types, DPX and w/o, the %RLN volumetric increase over baseline and the ratio of RLN/LLN were strong predictors of successful tumor suppression (LLN is left inguinal LN). The area under the curve (AUC) was greatest, between 0.75-0.85, two (%RLN) or three (RLN/LLN) weeks post-vaccination. For optimized critical thresholds we found these biomarkers consistently had sensitivity >90% and specificity >70% indicating strong prognostic potential. Vaccination with DepoVax had a more pronounced effect on draining lymph nodes than w/o emulsion vaccines, which correlated with a higher anti-tumor activity in DPX-treated mice.

Using MRI cell tracking to monitor immune cell recruitment in response to a peptide-based cancer vaccine: Tracking Immune Cells in Response to Immunotherapy Using MRI

MRI cell tracking can be used to monitor immune cells involved in the immunotherapy response, providing insight into the mechanism of action, temporal progression of tumor growth, and individual potency of therapies. To evaluate whether MRI could be used to track immune cell populations in response to immunotherapy, CD8+ cytotoxic T cells, CD4+CD25+FoxP3+ regulatory T cells, and myeloid‐derived suppressor cells were labeled with superparamagnetic iron oxide particles.